Hypertrophic cardiomyopathy dysfunction mimicked in human engineered heart tissue and improved by sodium–glucose cotransporter 2 inhibitors

Figures

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  Figure 1. Contractile phenotype and Empa treatment of spontaneously beating R403Q EHTs. (A) Representative picture of a human EHT as viewed by the video camera and evaluated by automated figure recognition (squares). (B) Schematic contraction peak with evaluated parameters of contractile function. (C) Examples of spontaneous beating patterns of R403Qic and R403Q EHT after 6 weeks of culture (red line, original recording; pink line, velocity). (D) Example of beating patterns of an R403Q EHT at baseline and after Empa treatment. Dotted lines allow us to compare the Empa effect on relaxation to baseline. (E) Effect of cumulative treatment with Empa for 1 h on contractility in starvation medium in R403Qic (n = 11, 39 ± 1 days) and R403Q (n = 12, 46 ± 3 days). (F) Comparison of the Empa (1 µM) effect between R403Qic and R403Q, calculated as the change in force or T280% as a percentage compared to baseline. Data are expressed as mean ± SEM (E, F; 2–3 independent EHT preparations). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Baseline (E) or R403Qic (F), repeated measures one-way ANOVA followed by Dunnett’s post-test (E) or unpaired Student’s t-test (F).

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  Figure 2. Contractile phenotype and Empa treatment of electrically stimulated R403Q EHTs. (A) Examples of beating patterns of an electrically stimulated (1 Hz) R403Qic and R403Q EHT (red line, original recording; pink line, and velocity). (B) EHT width, force of contraction and T280% of R403Qic (n = 24, 42 ± 3 days) were compared to R403Q (n = 24, 42 ± 0.2) EHTs paced at 1 Hz in starvation medium. (C) Example of beating patterns of an electrically stimulated (1 Hz) R403Q EHT at baseline and after Empa treatment. Dotted lines allow us to compare the Empa effect on relaxation to baseline. (D) Effect of Empa treatment for 1 h on contractility in starvation medium in R403Qic (n = 10, 39 ± 3 days) and R403Q (n = 9, 43 ± 0.2 days) paced at 1 Hz. (E) Comparison of the Empa (1 µM) effect on contractility between R403Qic and R403Q, calculated as the change compared to baseline. Data are expressed as mean ± SEM (B; three independent EHT preparations, D, E; two independent EHT preparations). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. R403Qic (B, E) or Baseline (D), unpaired Student’s t-test (B, E) or Repeated measures one-way ANOVA followed by Dunnett’s post-test (D).

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  Figure 3. Comparison of SGLT2i-effects on contractility in EHT. (A) Effect of Cana treatment for 1 h on force and T280% in spontaneously beating R403Q (n = 9, 52 ± 5 days). At 10 µM Cana, 3 EHTs stopped beating. (B) Effect of Dapa treatment for 1 h in spontaneously beating R403Q (n = 8, 48 ± 5 days). At 50 µM Dapa, 4 EHTs stopped beating. (C) Empa, Cana, and Dapa effects on T280% in spontaneously beating (Spon) and in paced (1 Hz) R403Q were compared to each other, calculated as the change in T280% as a percentage compared to baseline (Empa data from Figures 1F and 2E). (D) The effect size on relaxation time of Empa and Cana was compared between R403Qic and R403Q, showing a significantly larger reduction in relaxation time upon Empa and Cana treatment in R403Q compared to R403Qic (Empa data from Figures 1F and 2E, Cana data in R403Qic from Supplementary material online, Figure S8B). Data are expressed as mean ± SEM (A: two independent EHT preparations, B: one EHT preparation). (A, B) #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Baseline or (D) R403Qic, (A, B) a mixed-effects analysis followed by Dunnett’s post-test was performed if values are missing because EHTs stopped beating as a result of…

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  Figure 4. SGLT2i-effect on relaxation time in R403Q EHT depends on culture duration. (A) Western blots of R403Q EHTs stored on different days after EHT casting, as indicated above each lane. A human donor sample (donor) was loaded as a positive control for cardiac troponin I (cTnI), a mouse soleus sample as a positive control for slow skeletal troponin I (ssTnI), and HEK 293 cells as a negative control for TnI. cTnI expression corrected for actin increased upon prolonged culture time of R403Q EHT. This was also demonstrated with an antibody that detects both cTnI and ssTnI. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α-actinin staining illustrate that similar amounts of protein were loaded in each lane. The cTnI blot was cut at ∼50 kDa and ∼100 kDa, and the cTnI-ssTnI blot was cut at ∼85 kDa and ∼43 kDa. (B) Spontaneously beating R403Q were treated with SGLT2i (n = 3–4 EHTs per SGLT2i) on different days after EHT casting with cumulative concentrations for 1 h. Measurements with Empa, Cana, and Dapa were performed on days 20, 28, 37, 55, and 69, and with Cana also on day 44. The effect of SGLT2i on T280% was expressed as SGLT2i-induced changes compared to the baseline expressed in…

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  Figure 5. Empa and Cana effects on Ca2+ handling and contractility in 2D MYH7-R403Q and TNNT2-R92Q hiPSC-CM. R403Qic and R403Q (∼30–32 days old) hiPSC-CM were thawed and cultured in 2D and after 4 weeks contraction kinetics and Ca2+ handling were measured simultaneously in Tyrode medium. Relaxation time (T280%), Ca2+ transient decay time (Ca2+ decay time), and Ca2+ transient amplitude (Ca2+ amplitude) were measured. (A) R403Qic and R403Q hiPSC-CM treated for 15 min with 1 or 10 µM Empa. (B) R403Q hiPSC-CM treated for 15 min with 1, 3, or 10 µM Cana. (C) Contractile parameters of isogenic control hiPSC-CM (R92Qic) were compared to hiPSC-CM harbouring a TNNT2-R92Q mutation (R92Q) 35–45 days after thawing (60 days old). (D) R92Q were treated for 15 min with 10 µM Cana. The Ca2+ amplitude could only be determined as a relative change compared to the baseline. Each data point represents the average value of at least three spots measured within one well. Data are expressed as mean ± SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. (A, B, D) Baseline or (C) R92Qic, and (A–D) unpaired Student’s t-test.

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  Figure 6. Potential molecular targets of SGLT2i investigated in R403Q EHT. (A) Empa treatment for 1 h lowers relaxation time of spontaneously beating R403Q (n = 12, 70 ± 10 days) in starvation medium without glucose and insulin. (B) Effect of treatment with the selective NHE-1 inhibitor cariporide (Cari) for 1 h on contractility in R403Q (n = 9, 61 ± 7 days). (C) Treatment with the late Na+ current inhibitor ranolazine hydrochloride (Ran) for 1 h in spontaneously beating R403Q (1–50 µM: n = 6, 54 ± 6 days). (D) Effect of treatment with the mitochondrial antioxidant elamipretide (SS-31; n = 7, 79 ± 0 days) for 1 h on contractility in R403Q. (E, F) Western blots of R403Q treated for 1 h with DMSO (D) or Empa (1 or 50 µM). As control samples a human donor sample (donor) was loaded and an untreated or isoprenalin (Iso, 100 nM) treated control EHT (Ctrl). Almost no phosphorylation of PLB at Ser16 or cMyBP-C at Ser282 was detected in R403Q, in contrast to control samples. Results were identical when GAPDH or α-actinin was used as a loading control (indicated by α-actinin/GAPDH). Blots were cut at 75 kDa and below 20 kDa. Data are expressed as mean ± SEM (A: 3 independent EHT preparations; B, C:…

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  Figure 7. Molecular targets of SGLT2i that enhance relaxation in R403Q EHT. (A) Treatment with the L-type Ca2+ channel blocker diltiazem hydrochloride (Dilt) for 1 h in spontaneously beating R403Q (n = 10, 90 ± 1 days) and Ctrl (n = 8, 82 ± 3 days). R403Q EHTs stopped beating at 1 µM Dilt (three EHTs) and 3 µM Dilt (six EHTs). (B) The binding affinity of Cana for the dihydropyridine site of the L-type Ca2+ channel was determined (two replicates) in the rat cerebral cortex via a radioligand binding assay. Log(inhibitor) vs. response variable slope (four parameters) was fitted with the constraint top is 100. The concentration causing a half-maximal inhibition of control specific binding (IC50) was 37 µM. (C) Treatment with the potent NCX inhibitor SEA0400 for 1 h lowered relaxation time of spontaneously beating R403Q (n = 6, 56 ± 8 days), whereas SEA0400 increased relaxation time in Ctrl (n = 7, 81 ± 2 days). Treatment with SEA0400 resulted in some EHTs in irregular beating patterns (R403Q: 1 EHT at 33 nM; Ctrl: 1 EHT at 100 nM), which were excluded from data analyses. (D) Examples of beating patterns of a Ctrl and R403Q EHT with the same beating frequency, before treatment (baseline) and…

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  Figure 8. Effects of Cana on APs and membrane currents in TNNT2-R92Q hiPSC-CM. (A) Typical APs in the absence and the presence of 10 µmol/L Cana and average effects (depicted as dot plots) of 1, 3, and 10 µmol/L Cana on AP parameters. MDP = maximum diastolic potential, APA = maximal AP amplitude, Vmax = maximal AP upstroke velocity, and APD20, APD50, APD90 = AP duration at 20, 50, and 90% of repolarization. (B) Typical sodium currents (INa), evoked by 300 ms depolarizing pulses from −120 to −20 mV, in the absence and the presence of 10 µmol/L Cana. The dot plots show the average effects on peak and late INa. (C) Current–voltage (I–V) relationships of the Na+/Ca2+ exchange current (INCX) in the absence and the presence of 10 µmol/L Cana. INCX was measured during a descending ramp as a NiCl2-sensitive current. The dot plots indicate that INCX was significantly affected by Cana, with a more pronounced reduction at +70 mV, i.e. reverse-mode of INCX, than at −120 mV, i.e. the forward mode (D). Typical L-type Ca2+ current (ICa,L), evoked by 500 ms depolarizing pulses from −60 to 0 mV, in the absence and the presence of 10 µmol/L Cana. Neither the amplitude nor the time constant (τ) of current…